Using the gene for bovine pancreatic trypsin inhibitor (BPTI), a small, well-characterized protein for which the folding pathway has largely been elucidated, and site-specific mutagenesis, a variety of BPTI mutant proteins will be created, with the mutations primarily within the first 20 residues of the chain. Adapting the procedure of Creighton and co-workers, the folding pathway of those mutant BPTIs which exhibit reversible denaturation will be determined by following the refolding of reduced, denatured mutant BPTIs using high-performance hydrophobic interaction chromatography. By computer simulation of these results, the relevant rate constants and activation energies will be determined. From these data, the free energy surface for the folding of the peptide chain will be calculated. Concurrently, molecular mechanics calculations on unfolded forms will be made to determine unfolded and partially folded structures consistent with the experimental free energy surface. These experiments will provide insight into how the amino acid sequence alters the folding pathway, and subsequently the final, folded protein, thus serving to further under-standing of such phenomena as antigenicity, enzymatic activity, membrane transport, receptor activity, etc.